Modulation of Monocyte-Driven Myositis in Alphavirus Infection Reveals a Role for CX ₃ CR1 ⁺ Macrophages in Tissue Repair

Abstract

Arthritogenic alphaviruses such as Ross River and Chikungunya viruses cause debilitating muscle and joint pain and pose significant challenges in the light of recent outbreaks. How host immune responses are orchestrated after alphaviral infections and lead to musculoskeletal inflammation remains poorly understood. Here, we show that myositis induced by Ross River virus (RRV) infection is driven by CD11b hi Ly6C hi inflammatory monocytes and followed by the establishment of a CD11b hi Ly6C lo CX 3 CR1 ϩ macrophage population in the muscle upon recovery. Selective modulation of CD11b hi Ly6C hi monocyte migration to infected muscle using immune-modifying microparticles (IMP) reduced disease score, tissue damage, and inflammation and promoted the accumulation of CX 3 CR1 ϩ macrophages, enhancing recovery and resolution. Here, we detail the role of immune pathology, describing a poorly characterized muscle macrophage subset as part of the dynamics of alphavirus-induced myositis and tissue recovery and identify IMP as an effective immunomodulatory approach. Given the lack of specific treatments available for alphavirus-induced pathologies, this study highlights a therapeutic potential for simple immune modulation by IMP in infected individuals in the event of large alphavirus outbreaks. IMPORTANCE Arthritogenic alphaviruses cause debilitating inflammatory disease, and current therapies are restricted to palliative approaches. Here, we show that following monocyte-driven muscle inflammation, tissue recovery is associated with the accumulation of CX 3 CR1 ϩ macrophages in the muscle. Modulating inflammatory monocyte infiltration using immune-modifying microparticles (IMP) reduced tissue damage and inflammation and enhanced the formation of tissue repair-associated CX 3 CR1 ϩ macrophages in the muscle. This shows that modulating key effectors of RESULTSAcute RRV-induced myositis is followed by recovery and tissue repair. To assess the kinetics of muscle tissue inflammation and repair following RRV infection, C57BL/6 (wild-type [WT]) mice were infected subcutaneously with 10 4 PFU of the mouse virulent RRV T48 strain as described previously (24,25). Mice were scored according to clinical manifestations from the onset of hind limb dysfunction at 6 or 7 days postinfection (dpi), to the acute phase at 9 or 10 dpi with severe hind limb dysfunction, lethargy, and muscle tissue damage (Fig. 1A). During the acute phase, mice displayed moderate-tosevere motor impairment, were unable to walk or stand on their hind legs, and often dragged their hind legs when moving. From 13 to 15 dpi, mice regained hind limb function and progressed toward full recovery, approximately around 15 to 16 dpi.FIG 1 RRV-induced, mononuclear phagocyte-driven myositis leads to severe muscle damage followed by muscle tissue recovery. (A) RRV disease score in 21-day-old C57BL/6 mice infected with RRV T48 (10 4 PFU s.c.) or mock infected with PBS. Mice were monitored daily for signs of musculoskeletal dysfunction and loss of hind limb function. Data are means...

Muscle Tissue Damage and Recovery After EV71 Infection Correspond to Dynamic Macrophage Phenotypes

“…The signals regulating interchangeable macrophage phenotypes remain to be investigated. Of interest, our results on dynamic macrophages phenotypes changes in EV71-induced myositis is reminiscent of the recent publication, which showed that myositis induced by Ross River virus (an Arthritogenic alphaviruses) infection is driven by CD11b hi Ly6C hi inflammatory monocytes followed by the establishment of CD11b hi Ly6C low/macrophages for the recovery of damaged muscles (41). It is plausible that, during viral infection, the muscle tissue damage and subsequent repair seem associated with the differentially producing various cytokines/soluble factors to regulate the macrophage phenotype for eradicating and resolving infection.…”

Altered Spatial and Temporal Gait Parameters in Mice Infected with Ross River Virus

“…That these temporal gait parameters were modified early during the period of peak disease suggests that acute RRV disease most significantly impacts gait during the onset of disease. Accordingly, the onset period of the disease (6 to 9 dpi) in RRV-infected mice is associated with peak expression of proinflammatory mediators, a sharp influx of inflammatory monocytes, and myositis in skeletal muscle ( 14 ). Known changes in the immunopathology of the joints and muscles are likely to contribute to gait modifications in RRV-infected mice.…”

“…Thus, it is unclear whether overt signs of disease are required for the DigiGait system to detect significant changes in gait or whether more severe disease results in changes in gait that are detected by DigiGait. It is important, then, to note that changes in gait in RRV-infected mice were detected only at peak disease, when clinical signs of disease are most obvious and levels of markers of immunopathogenesis are at their highest ( 14 ).…”

“…Disease signs peak at approximately 10 dpi with severe hindlimb dysfunction, loss of grip strength, and severe weight loss. The RRV-induced inflammatory response results in severe myositis and bone loss in mice at peak disease ( 13 , 14 ). At 15 to 21 dpi, the disease begins to resolve, with mice regaining hindlimb function and gaining weight.…”

New Insights into Chikungunya Virus Infection and Pathogenesis

“…Instead, it results in attenuated muscle damages reflected by diminished necrosis and local induction of IL-6, IL-1β, TNFα, and IP10, which are biomarkers of disease severity in humans and mice (44,46). Besides tissue damage, the attraction of infiltrating monocytes/macrophages is also critical to local inflammation and viral persistence (39,(47)(48)(49). More specifically, infection of fibroblast-like synoviocytes results in the secretion of IL-6, IL-8, and CCL2, which attracts phagocytes.…”